Optical Detection Technologies for Waterborne Pathogens - Waterborne Pathogens: Detection Methods and Applications - page 111

as being included in biosensor reviews. This is particularly problematic for
“indirect” optical biosensors, where detection occurs via a secondary label.
Therefore, in some cases, work is referred to in both chapters.
The chapter first deals with optical detection techniques requiring sam-
ple labeling, mainly fluorescence (Section 5.1 ). A brief explanation of the
fluorescence process and different approaches is provided. An alternative
method for visualization of pathogens is the use of enzymes to generate a
colored precipitate. In this section, we also describe attempts to automate
the existing protocols, presented in Chapter 3, as well as general develop-
ments toward miniaturized microscopes.
Flow cytometry can utilize light scattering to discriminate between
pathogens based on size and shape in a label-free way. However, the com-
bination with fluorescence labeling offers greater selectivity and the ability
to detect viability and even species; therefore flow cytometry is presented
in Section 5.1 .
The next Section 5.2 deals with label-free technologies. In terms of
intrinsic, label-free methods, vibrational spectroscopy techniques, including
infrared absorption and Raman spectroscopy, are described in detail.
5.1. TECHNIQUES USING LABELING
5.1.1. Fluorescence
The use of fluorescence is a popular method of detection. The Jablon-
ski diagram below illustrates the fluorescence process ( Fig. 5.1 ). When a
fluorophore is illuminated with light, absorbance of a photon of sufficient
energy results in excitation of an electron from the ground state into higher
electronic energy states. Various means are available to that electron to
relax, lose the excess energy and return to the ground state; one of these is
fluorescence, by which a longer wavelength photon is remitted.
For a more detailed introduction to fluorescence and the different pro-
cesses which can occur, we recommend topics such as The Fundamentals of
Light Microscopy and Electronic Imaging ; for a basic introduction, websites with
online tutorials, such as the Invitrogen website, are useful and have good
animations ( Fig. 5.1 ).
However, there are some drawbacks to fluorescence detection, including
the additional costs of the fluorescent label, problems with photobleaching
and the challenges of obtaining specificity. Photobleaching is a process by
which the repeated cycling of a fluorophore between ground and excited